Three dimensional cell matrices are used in tissue engineering and biotechnology for in-vitro and in-vivo cell culturing. For example, the paper TREATMENT OF DEEP CARTILAGE DEFECTS IN THE KNEE WITH AUTOLOGOUS CHONDROCYTE TRANSPLANTATION, M Brittberg, A Lindahl, A Nilsson, C Ohlsson, O Isaksson, L Peterson, N Engl J Med, Oct. 6, 1994: 331(14): 889-895 reports successful transplantation of healthy knee cartilage. The transplant was accomplished by obtaining healthy knee chondrocytes, culturing them in the laboratory for 14-21 days then injecting the cultured chondrocytes to the damaged area. The culturing method is typically monolayer or planar culturing as described, for example in the paper DEVELOPMENT OF A TISSUE ANALOG FOR CARTILAGE REPAIR, J. M. Pachence, S. R. Frenkel, and J. Lin, Mat. Res. Soc. Symp. Proc. Vol. 252, 1992, discusses culturing cells (chondrocytes) on a planar substrate and harvesting them for placement. In this paper, instead of direct in-vivo injection, placement was into a three dimensional solid scaffold of collagen. Nevertheless, the monolayer culturing was the same which turns cultured cells to undesirable "fibroblast-like cells".
Another paper, CHONDROCYTES EMBEDDED IN COLLAGEN GELS MAINTAIN CARTILAGE PHENOTYPE DURING LONG TERM CULTURES, T Kimura, N Yasui, S Ohsawa, K Ono, discusses successful chondrocyte phenotype in-vitro maintenance in a collagen gel. However, no discussion of cell recovery suggests that collagen gel is useful solely as a cell carrier for implantation or for studying cell function, but not for in-vitro propagation.
The book chapter 53 BIOMATERIALS INVOLVED IN CARTILAGINOUS IMPLANTS, Z. Nevo, D. Robinson, D. G. Mendes, N. Halperin, in the book Biomaterials Science, Eds: B D Rafner, A S Hoffman, F J Schoen, J E Lemons, Academic Press 1996 discusses using a hyaluronic-acid based adhesive gel embedded with chondrocyte cell culture in-vivo. The chapter further discusses collagen based scaffolds and identifies the limitation that the cells tend to grow mainly on the surface of the collagen scaffold because of lack of nourishment of more deeply situated cells. Polylactide and polyglycolide matrices are also discussed concluding that further investigation is needed. These bioabsorbable materials are solely used as scaffolds for cell seeding and later implantation with no discussion of in-vitro propagation or recovery of cells from the scaffolds.
Gelling three dimensional matrices have also been investigated as reported in DEDIFFERENTIATED CHONDROCYTES REEXPRESS THE DIFFERENTIATED COLLAGEN PHENOTYPE WHEN CULTURED IN AGAROSE GELS, P D Benya, J D Shaffer, Cell Aug. 30, 1982 (1): 215-224. Agarose culture is a method to maintain chondrocyte phenotype in a three dimensional matrix. However, the cells in agarose are not readily recoverable. Agarose may be liquefied by heating to 80.degree. C., but that overheats the cells and the cells do not survive. Alternatively, the agarose may be degraded with the enzyme agarase. However, the effect of the agarase is insufficient to readily dissolve the bulk gel and collect the cells.
Another gelling three-dimensional cell matrix has the trade name Matrigel which is naturally produced by tumor cells. Enzymatic action is required for dissolution or degradation of the gel which, again damages any cells therein. Because Matrigel is a tumor cell product, it is used primarily for in-vivo promotion of tumor cell growth for mice studies as exemplified in the paper ENHANCED TUMOR GROWTH OF BOTH PRIMARY AND ESTABLISHED HUMAN AND MURINE TUMOR CELLS IN ATHYMIC MICE AFTER COINJECTION WITH MATRIGEL, R Fridman, M C Kibbey, L S Royce, M Zain, M Sweeney, D L D L Jicha, J R Yannelli, G R Martin, H K Kleinman, J Natl Cancer Inst Jun. 5, 1991: 83(11): 769-774.
The paper FIBROBLAST GROWTH FACTOR STIMULATED COLONY FORMATION OF DIFFERENTIATED CHONDROCYTES IN SOFT AGAR, Y Kato, M Iwamoto, T Koike, J Cell Physol December 1987: 133(3): 491-498, discusses a soft agar as a three dimensional cell matrix wherein the fibroblast growth factor selectively stimulates growth of differentiated chondrocytes in-vitro. It is believed that cells are not easily recovered from soft agar, therefore, making soft agar useful for studying cell function but not useful for in-vitro cell propagation for subsequent implantation.
The paper PHENOTYPE AND BIOLOGICAL ACTIVITY OF NEONATAL EQUINE CHONDROCYTES CULTURED IN A THREE-DIMENSIONAL FIBRIN MATRIX, D A Hendrickson, A J Nixon, H N Erb, G Lust, Am J Vet Res March 1994, 55(3): 410-414, discusses using a3D culture in fibrin to study the differentiation and function of embedded chondrocytes. Because fibrin can not be readily dissolved by any means to release the embedded cells, fibrin is not a candidate for in-vitro cell propagation.
The paper PHENOTYPE STABILITY OF BOVINE ARTICULAR CHONDROCYTES AFTER LONG-TERM CULTURE IN ALGINATE BEADS, H J Hauselmann, R J Fernandez, S S Mok, T M Schmid, J A Block, M B Aydelotte, K E Kuettner, E J Thonar, J Cell Sci, January 1994: 107(Pt 1): 17-27, reports chondrocyte phenotype stability with the ability to recover cells by chelating the calcium. Although useful either as a carrier for cell implantation or for studying cell function or behavior, alginate beads are not attractive for in-vitro propagation because of the additional steps of chelation and separation of cells from the chelator.
Use of a reverse thermosensitive polymer has been demonstrated for in-vivo cartilage formation. The reverse thermosensitive polymer was Pluronics, a co-polymer of polyethylene oxide and polypropylene oxide which is biodegradable, biocompatible, exists as a liquid at room temperature and polymerizes to a thick gel when exposed to physiologic temperature (body temperature). This work has been reported in two abstracts (1) INJECTABLE CARTILAGE USING THERMOSENSITIVE POLYMERS: A POTENTIAL METHOD FOR NIPPLE RECONSTRUCTION, Y I Cao, C Peetz, K Tran, C A Vacanti, Tissue Engineering Society, Dec. 12-13, 1996, Orlando, Fla., and (2) FATE OF INJECTABLE CHONDROCYTE/POLYMER CONSTRUCTS IN IMMUNOCOMPETENT ANIMAL, S K Ashiku, M A Randolph, D J Mathisen, M J Yaremchuk, C A Vacanti, Tissue Engineering Society, Dec. 13-15, 1996, Orlando, Fla. Although used for implantation, the Pluronics material was not used for in-vitro cell propagation. Disadvantages include the high concentration of the Pluronics material (30-40% w/v) and possibly difficult recovery.
In spite of the forgoing research, the standard method of cell implantation is by propagation in a monolayer culture followed by injection. Until the present invention, there has simply not been a sufficiently reliable method of 3D cell-seeding and recoverable in-vitro propagation for cell propagation.
Accordingly, there is a need in the field of three dimensional cell matrices for a three dimensional cell matrix useful for in-vitro cell propagation from which the cells may be separated without damage, without added compounds (e.g. chelator), and without excessive amount of matrix material to be removed, wherein phenotypical cells may be recovered with their original phenotype and subsequently used for cell seeding and implantation.